The unifying goal of this proposed research is to elucidate and characterize the proton transfer processes occurring during the hydration of carbon dioxide when catalyzed by carbonic anhydrase. The maximal catalytic hydration rate is so large as to require proton transfer between buffers in solution and the enzyme active site, a process which has not yet been demonstrated in any other enzyme-catalyzed reaction. The bimolecular rate constant for proton transfer from buffers in solution to the active site of several isozymes of carbonic anhydrase will be determined using oxygen-18 exchange and stopped-flow techniques. Such bimolecular rate constants for many different buffers will be measured to determine whether the proton transfer between buffer and active site follows the Bronsted catalysis law and whether binding of buffers to the enzyme is involved. These results will be compared with rate constants for proton transfer obtained by fluorescence techniques. Measuring proton transfer from the excited state of fluorescent buffers to residues in the vicinity of the active site, we will evaluate the possibility that a proton shuttle mechanism exists to enhance proton transfer between the enzyme active site and solution. The utilization of this transferred proton in the catalytic pathway will be investigated by deuterium solvent isotope effects and the rate of exchange of water between the active site and solvent water will be determined by 18O- and 13C-isotope exchange kinetics.